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United States Patent |
5,693,775
|
Nathans
,   et al.
|
December 2, 1997
|
Fibroblast growth factor homologous factor-1 (FHF-1) and methods of use
Abstract
A novel protein, fibroblast growth factor homologous factor-1 (FHF-1), the
polynucleotide sequence encoding FHF-1, and the deduced amino acid
sequence are disclosed. Also disclosed are diagnostic and therapeutic
methods of using the FHF-1 polypeptide and polynucleotide sequences and
antibodies which specifically bind to FHF-1.
Inventors:
|
Nathans; Jeremy (Baltimore, MD);
Smallwood; Philip M. (Woodbine, MD);
Macke; Jennifer P. (Columbia, MD)
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Assignee:
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The Johns Hopkins University School of Medicine (Baltimore, MD)
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Appl. No.:
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439725 |
Filed:
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May 12, 1995 |
Current U.S. Class: |
536/23.1; 435/252.33; 435/320.1; 435/369; 536/24.3 |
Intern'l Class: |
C07H 021/04 |
Field of Search: |
536/22.1,23.1,24.3
530/350
|
References Cited
Other References
Zhan, et al., The Human FGF-5 Oncogene Encodes a Novel Protein related to
Fibroblast Growth Factors, Molecular and Cellular Biology, 8(8):3487,
1988.
Miyamoto, et al., Molecular Cloning of a Novel Cytokine cDNA Encoding the
Ninth Member of the Fibroblast Growth Factor Family, Which Has a Unique
Secretion Property, Molecular and Cellular Biology, 13(7):4251, 1993.
Payson, et al., Cloning of two novel forms of human acidic fibroblast
growth factor (aFGF) mRNA, Nucleic Acids Research, 21(3):489, 1993.
Yoshida, et al., Genomic sequence of hst, a transforming gene encoding a
protein homologous to fibroblast growth factors and the int-2-encoded
protein, Proc. Natl. Acad. Sci. USA, 84:7305, 1987.
Brookes, et al., The mouse homologue of hst/ k-FGF: sequence, genome
organization and location relative to int-2, Nucleic Acids Research,
17(11):4037, 1989.
Iida, et al., Human hst-2 (FGF-6) oncogene: cDNA cloning and
characterization, Oncogene, 7:303, 1992.
Merlo, et al., The Mouse int-2 gene Exhibits Basic Fibroblast Growth Factor
Activity in a Basic Fibroblast Growth Factor-responsive Cell Line, Cell
Growth & Differentiation, 1(10):463, 1990.
Davis et al. Basic Methods in Molecular Biology, pp. 51-57 Elsevier Science
Publishing Co., New York, NY (1986).
|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Whisenant; Ethan
Attorney, Agent or Firm: Fish & Richardson P.C.
Claims
We claim:
1. An isolated polynucleotide sequence that encodes an FHF-1 polypeptide
having a molecular weight of about 30 kD as determined by SDS-PAGE; and
having the amino acid sequence of SEQ ID NO: 2.
2. The polynucleotide of claim 1, wherein the FHF-1 nucleotide sequence is
selected from the group consisting of:
(a) SEQ ID NO:1, wherein T can also be U;
(b) nucleic acid sequences complementary to SEQ ID NO: 1;
(c) fragments of (a) or (b) that are at least 15 bases in length and that
will selectively hybridize to DNA which encodes the amino acid sequence of
SEQ. ID NO:2, under stringent conditions.
3. The polynucleotide sequence of claim 1, wherein the polynucleotide is
isolated from a mammalian cell.
4. The polynucleotide of claim 3, wherein the mammalian cell is a human
cell.
5. An expression vector which contains in operable linkage the
polynucleotide of claim 1.
6. The vector of claim 5, wherein the vector is a plasmid.
7. The vector of claim 5, wherein the vector is a virus.
8. A host cell stably transformed with the vector of claim 5.
9. The host cell of claim 8, wherein the cell is prokaryotic.
10. The host cell of claim 8, wherein the cell is eukaryotic.
11. The polynucleotide of claim 1, which has the sequence of SEQ ID NO:1.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to growth factors and specifically to a
novel member of the fibroblast growth factor family, denoted fibroblast
growth factor homologous factor-1 (FHF-1) and the polynucleotide encoding
FHF-1.
2. Description of Related Art
The fibroblast growth factor family encompasses a group of structurally
related proteins with a wide range of growth promoting, survival, and/or
differentiation activities in vivo and in vitro (reviewed in Baird, A.,
and Gospodarowicz, D. Ann N.Y. Acad. Sci. 638: 1, 1991; Eckenstein, F. P.,
J. Neurobiology 25: 1467, 1994; Mason, I. J. Cell 78: 547, 1994). As of
December 1994, nine members of this family had been characterized by
molecular cloning. The first two members of the family to be
characterized, acidic fibroblast growth factor (aFGF/FGF-1) and basic
fibroblast growth factor (bFGF/FGF-2), have been found in numerous
tissues, including for example brain, eye, kidney, placenta, and adrenal
(Jaye et al., Science 233: 541, 1986; Abraham et al., Science 233: 545,
1986). These factors have been shown to be potent mitogens and survival
factors for a variety of mesoderm and neurectoderm-derived tissues,
including fibroblasts, endothelial cells, hippocampal and cerebral
cortical neurons, and astroglia (Burgess, W. H. and Maciag, T., Ann. Rev.
Biochemistry 58: 575, 1989). Additional members of the FGF family include:
i-nt-2/FGF-3, identified as one of the frequent sites of integration of
the mouse mammary tumor virus, and therefore a presumptive oncogenic
factor (Smith et al., EMBO J. 7: 1013, 1988); FGF-4 (Delli-Bovi et al.,
Cell 50: 729, 1987) and FGF-5 (Zhan et al., Mol. Cell Biol.8: 3487, 1988)
as transforming genes in the NIH 3T3 transfection assay; FGF-6, isolated
by molecular cloning based on its homology to FGF-4 (Marics et al.,
Oncogene 4: 335 (1989); keratinocyte growth factor/FGF-7, identified as a
mitogen for keratinocytes (Finch et al., Science 245: 752, 1989); FGF-8 as
an androgen-induced mitogen for mammary carcinoma cells (Tanaka et al.,
Proc. Natl. Acad. Sci. USA 89: 8928, 1992); and FGF-9 as a mitogen for
primary astrocytes (Miyamoto et al., Mol. Cell Biol. 13: 4251, 1993).
Several of the FGFs, including aFGF and bFGF, lack a classical signal
sequence; the mechanism by which they are secreted is not known.
All members of the FGF family share approximately 25% or more amino acid
sequence identity, a degree of homology indicating that they are likely to
share nearly identical three-dimensional structures. Support for this
inference comes from a comparison of the three-dimensional structures of
bFGF and interleukin 1-beta determined by x-ray diffraction (Eriksson et
al., Proc. Natl. Acad. Sci USA 88: 3441, 1991; Zhang et al., Proc. Natl.
Acad. Sci USA 88: 3446, 1991; Ago et al., J. Biochem. 110: 360, 1991).
Although these proteins share only 10% amino acid identity, the alpha
carbon backbones of the two crystal structures can be superimposed with a
root-mean square deviation of less than 2 angstroms (Zhang et al., Proc.
Natl. Acad. Sci USA 88: 3446, 1991). Both proteins consist almost entirely
of beta-sheets, which form a barrel composed of three copies of a
four-stranded beta-meander motif. The likely heparin- and receptor-binding
regions are located on nearby regions on one face of the protein.
aFGF, bFGF, and FGF-7/KGF have been shown to exert some or all of their
biological activity through high affinity binding to cell surface tyrosine
kinase receptors (e.g., Lee, P. L., et al., Science 245: 57, 1989;
reviewed in Johnson, D. E. and Williams, L. T., Adv. Cancer Res. 60: 1,
1993). Many members of the FGF family also bind tightly to heparin, and a
terniary complex of heparin, FGF, and transmembrane receptor may be the
biologically relevant signalling species. Thus far four different genes
have been identified that encode receptors for FGF family members. Recent
work has shown that receptor diversity is increased by differential mRNA
splicing within the extracellular ligand binding domain, with the result
that multiple receptor isoforms with different ligand binding properties
can be encoded by the same gene (Johnson, D. E. and Williams, L. T., Adv.
Cancer Res. 60: 1, 1993). In tissue culture systems, the binding of aFGF
or bFGF to its cell surface receptor activates phospholipase C-gamma
(Burgess, W. H. et al., Mol. Cell Biol. 10: 4770, 1990), a pathway known
to integrate a variety of mitogenic signals.
Identification and characterization of new members of the FGF family will
provide insights into the mechanisms by which cells and organs control
their growth, survival, senescence, differentiation, and recovery from
injury.
SUMMARY OF THE INVENTION
The present invention provides a cell growth, survival or differentiation
factor, FHF-1 and a polynucleotide sequence which encodes the factor. This
factor is involved in the growth, survival, and or differentiation of
cells within the central nervous system (CNS) as well as in peripheral
tissues.
The invention provides a method for detecting alterations in FHF-1 gene
expression which are diagnostic of neurodegenerative or neoplastic
disorders. In another embodiment, the invention provides a method for
treating a neurodegenerative or neoplastic disorder by modulating the
expression or activity of FHF-1.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide and predicted amino acid sequence of human
FHF-1.
FIG. 2 shows the alignment of the amino acid sequence of human FHF-1 and
each of the other nine members of the FGF family. Conserved residues are
highlighted. The FGF family members are: aFGF/FGF-1 (Jaye et al., Science
233: 541, 1986), bFGF/FGF-2 (Abraham et al., Science 233: 545, 1986),
int-2/FGF-3 (Smith et al., EMBO J. 7:1013, 1988), FGF-4 (Delli-Bovi et
al., Cell 50: 729, 1987), FGF-5 (Zhan et al., Mol. Cell Biol. 8: 3487,
1988), FGF-6 (Marics et al., Oncogene 4: 335, 1989); keratinocyte growth
factor/FGF-7 (Finch et al., Science 245: 752, 1989), FGF-8 (Tanaka et al.,
Proc. Natl. Acad. Sci. USA 89: 8928, 1992), and FGF-9 (Miyamoto et al.,
Mol. Cell Biol. 13: 4251, 1993).
FIG. 3 shows a dendrogram in which the length of each path connecting any
pair of FGF family members is proportional to the degree of amino acid
sequence divergence of that pair.
FIG. 4 shows that the gene encoding FHF-1 is located on human chromosome 3.
The human specific hybridization is found on chromosome 3.
FIG. 5 shows the production of FHF-1 in transfected human embryonic kidney
cells. Lanes 1, 3, and 5, total cell protein; lanes 2, 4, and 6 protein
present in the medium (secreted protein). Lanes 1 and 2, transfection with
cDNA encoding human growth hormone; lanes 3 and 4, transfection with cDNA
encoding FHF-1; lanes 5 and 6, transfection with cDNA encoding a novel
surface receptor fused to an immunoglobulin constant region. Protein
standards are shown to the left; from top to bottom their molecular masses
are 220, 97, 66, 46, 30, 21.5, and 14.3 kD.
FIG. 6 shows the tissue specificity of FHF-1 expression. Ten micrograms of
total RNA from the indicated mouse tissues was prepared (Chomczinski &
Sacchi. Anal. Biochem. 162: 156, 1987) and used for RNAse protection
(Ausabel et al., Current Protocols in Molecular Biology; New York: Wiley
Interscience, 1987) with a mouse FHF- 1 antisense probe that spanned 212
bases of exon 1 and the adjacent 100 bases of intron 1. RNAse protection
at the size expected for the 212 base exon 1 region of the probe
(arrowhead) was observed with RNA from brain, eye, and testis.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a growth factor, FHF-1, and a polynucleotide
sequence encoding FHF-1. FHF-1 is expressed at high levels in brain, eye
and testes tissues. In one embodiment, the invention provides a method for
detection of a cell proliferative disorder of central nervous system or
testes origin which is associated with FHF-1 expression or function. In
another embodiment, the invention provides a method for treating a cell
proliferative or immunologic disorder by using an agent which suppresses
or enhances FHF-1 expression or activity.
The structural homology between the FHF-1 protein of this invention and the
members of the FGF family, indicates that FHF-1 is a new member of the
family of growth factors. Based on the known activities of many of the
other members, it can be expected that FHF-1 will also possess biological
activities that will make it useful as a diagnostic and therapeutic
reagent.
Many growth factors have expression patterns or possess activities that
relate to the function of the nervous system. For example, one growth
factor in the TGF family, namely GDNF, has been shown to be a potent
neurotrophic factor that can promote the survival of dopaminergic neurons
(Lin, et al., Science, 260:1130). Another family member, namely
dorsalin-1, is capable of promoting the differentiation of neural crest
cells (Basler, et al., Cell, 73:687, 1993). The inhibins and activins have
been shown to be expressed in the brain (Meunier, et al., Proc. Nat'l.
Acad. Sci., USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988),
and activin has been shown to be capable of functioning as a nerve cell
survival molecule (Schubert, et al., Nature, 344:868, 1990). Another TGF
family member, namely GDF-1, is nervous system-specific in its expression
pattern (Lee, Proc. Nat'l. Acad. Sci., USA, 88:4250, 1991), and certain
other family members, such as Vgr-1 (Lyons, et al., Proc. Nat'l. Acad.
Sci., USA, 86:4554, 1989; Jones, et al., Development, 111:581, 1991), OP-1
(Ozkaynak, et al., J. Biol. Chem., 267:25220, 1992), and BMP-4 (Jones, et
al., Development, 111:531, 1991), are also known to be expressed in the
nervous system.
The expression of FHF-1 in brain and eye suggests that FHF-1 may also
possess activities that relate to the function of the nervous system.
FHF-1 may have neurotrophic activities for various neuronal populations.
Hence, FHF-1 .may have in vitro and in vivo applications in the treatment
of neurodegenerative diseases, such as amyotrophic lateral sclerosis, or
in maintaining cells or tissues in culture prior to transplantation.
In a first embodiment, the present invention provides a substantially pure
fibroblast growth factor homologous factor-1 (FHF-1) characterized by
having a molecular weight of about 30 kD as determined by reducing
SDS-PAGE and having essentially the amino acid sequence of SEQ ID NO:2.
The term "substantially pure" as used herein refers to FHF-1 which is
substantially free of other proteins, lipids, carbohydrates or other
materials with which it is naturally associated. One skilled in the art
can purify FHF- 1 using standard techniques for protein purification. The
substantially pure polypeptide will yield a single major band on a
non-reducing polyacrylamide gel. The purity of the FHF-1 polypeptide can
also be determined by amino-terminal amino acid sequence analysis. FHF-1
polypeptide includes functional fragments of the polypeptide, as long as
the activity of FHF-1 remains. Smaller peptides containing the biological
activity of FHF-1 are included in the invention.
The invention provides polynucleotides encoding the FHF-1 polypeptide.
These polynucleotides include DNA, cDNA and RNA sequences which encode
FHF-1. It is understood that all polynucleotides encoding all or a portion
of FHF-1 are also included herein, as long as they encode a polypeptide
with FHF- 1 activity. Such polynucleotides include naturally occurring,
synthetic, and intentionally manipulated polynucleotides. For example,
FHF-1 polynucleotide may be subjected to site-directed mutagenesis. The
polynucleotide sequence for FHF-1 also includes antisense sequences. The
polynucleotides of the invention include sequences that are degenerate as
a result of the genetic code. There are 20 natural amino acids, most of
which are specified by more than one codon. Therefore, all degenerate
nucleotide sequences are included in the invention as long as the amino
acid sequence of FHF-1 polypeptide encoded by the nucleotide sequence is
functionally unchanged.
Specifically disclosed herein is a DNA sequence encoding the human FHF-1
gene. The sequence contains an open reading frame encoding a polypeptide
244 amino acids in length. The human FHF-1 inititiator methionine codon
shown in FIG. 1 at position 332-334 is the first ATG codon following the
in-frame stop codon at nucleotides 323-325; a good consensus ribosome
binding site (TGGCCATGG; Kozak, Nucleic Acids Res., 15: 8125, 1987) is
found at this position. The next methionine codon within the open reading
frame is encountered 86 codons 3' of the putative initiator methionine
codon. As observed for aFGF and bFGF, the amino-terminus of the primary
translation product of FHF-1 does not conform to the consensus sequence
for a signal peptide to direct cotranslational insertion across the
endoplasmic reticulum membrane. The FHF-1 sequence lacks potential
asn-X-ser/thr site for asparagine-linked glycosylation. Preferably, the
human FHF-1 nucleotide sequence is SEQ ID NO: 1 and the deduced amino acid
sequence is preferably SEQ ID NO:2.
The polynucleotide encoding FHF-1 includes SEQ ID NO:1 as well as nucleic
acid sequences complementary to SEQ ID NO: 1. A complementary sequence may
include an antisense nucleotide. When the sequence is RNA, the
deoxynucleotides A, G, C, and T of SEQ ID NO:1 is replaced by
ribonucleotides A, G, C, and U, respectively. Also included in the
invention are fragments of the above-described nucleic acid sequences that
are at least 15 bases in length, which is sufficient to permit the
fragment to selectively hybridize to DNA that encodes the protein of SEQ
ID NO:2 under physiological conditions. Specifically, the fragments should
hybridize to DNA encoding FHF-1 protein under stringent conditions.
The FGF family member most homologous to FHF-1 is FGF-9, which shares 27%
amino acid identity when aligned with 10 gaps. Minor modifications of the
FHF-1 primary amino acid sequence may result in proteins which have
substantially equivalent activity as compared to the FHF-1 polypeptide
described herein. Such proteins include those as defined by the term
"having essentially the amino acid sequence of SEQ ID NO:2". Such
modifications may be deliberate, as by site-directed mutagenesis, or may
be spontaneous. All of the polypeptides produced by these modifications
are included herein as long as the biological activity of FHF-1 still
exists. Further, deletion of one or more amino acids can also result in a
modification of the structure of the resultant molecule without
significantly altering its biological activity. This can lead to the
development of a smaller active molecule which would have broader utility.
For example, one can remove amino or carboxy terminal amino acids which
are not required for FHF-1 biological activity.
The FHF-1 polypeptide of the invention encoded by the polynucleotide of the
invention includes the disclosed sequence (SEQ ID NO:2) and conservative
variations thereof. The term "conservative variation" as used herein
denotes the replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine or methionine for another, or the substitution of one polar
residue for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the like. The
term "conservative variation" also includes the use of a substituted amino
acid in place of an unsubstituted parent amino acid provided that
antibodies raised to the substituted polypeptide also immunoreact with the
unsubstituted polypeptide.
DNA sequences of the invention can be obtained by several methods. For
example, the DNA can be isolated using hybridization techniques which are
well known in the art. These include, but are not limited to: 1)
hybridization of genomic or cDNA libraries with probes to detect
homologous nucleotide sequences, 2) polymerase chain reaction (PCR) on
genomic DNA or cDNA using primers capable of annealing to the DNA sequence
of interest, and 3) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features.
Preferably the FHF-1 polynucleotide of the invention is derived from a
mammalian organism, and most preferably from human. Screening procedures
which rely on nucleic acid hybridization make it possible to isolate any
gene sequence from any organism, provided the appropriate probe is
available. Oligonucleotide probes, which correspond to a part of the
sequence encoding the protein in question, can be synthesized chemically.
This requires that short, oligopeptide stretches of amino acid sequence
must be known. The DNA sequence encoding the protein can be deduced from
the genetic code, however, the degeneracy of the code must be taken into
account. It is possible to perform a mixed addition reaction when the
sequence is degenerate. This includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is preferably
performed on either single-stranded DNA or denatured double-stranded DNA.
Hybridization is particularly useful in the detection of cDNA clones
derived from sources where an extremely low mount of mRNA sequences
relating to the polypeptide of interest are present. In other words, by
using stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the target
DNA to that single probe in the mixture which is its complete complement
(Wallace, et al., Nucl. Acid Res., 9:879, 1981; Maniatis, et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989).
The development of specific DNA sequences encoding FHF-1 can also be
obtained by: 1) isolation of double-stranded DNA sequences from the
genomic DNA; 2) chemical manufacture of a DNA sequence to provide the
necessary codons for the polypeptide of interest; and 3) in vitro
synthesis of a double-stranded DNA sequence by reverse transcription of
mRNA isolated from a eukaryotic donor cell. In the latter case, a
double-stranded DNA complement of mRNA is eventually formed which is
generally referred to as cDNA.
Of the three above-noted methods for developing specific DNA sequences for
use in recombinant procedures, the isolation of genomic DNA isolates is
the least common. This is especially true when it is desirable to obtain
the microbial expression of mammalian polypeptides due to the presence of
introns.
The synthesis of DNA sequences is frequently the method of choice when the
entire sequence of amino acid residues of the desired polypeptide product
is known. When the entire sequence of amino acid residues of the desired
polypeptide is not known, the direct synthesis of DNA sequences is not
possible and the method of choice is the synthesis of cDNA sequences.
Among the standard procedures for isolating cDNA sequences of interest is
the formation of plasmid- or phage-carrying cDNA libraries which are
derived from reverse transcription of mRNA which is abundant in donor
cells that have a high level of genetic expression. When used in
combination with polymerase chain reaction technology, even rare
expression products can be cloned. In those cases where significant
portions of the amino acid sequence of the polypeptide are known, the
production of labeled single or double-stranded DNA or RNA probe sequences
duplicating a sequence putatively present in the target cDNA may be
employed in DNA/DNA hybridization procedures which are carried out on
cloned copies of the cDNA which have been denatured into a single-stranded
form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).
A cDNA expression library, such as lambda gt11, can be screened indirectly
for FHF-1 peptides having at least one epitope, using antibodies specific
for FHF-I. Such antibodies can be either polyclonally or monoclonally
derived and used to detect expression product indicative of the presence
of FHF-1 cDNA.
DNA sequences encoding FHF-1 can be expressed in vitro by DNA transfer into
a suitable host cell. "Host cells" are cells in which a vector can be
propagated and its DNA expressed. The term also includes any progeny of
the subject host cell. It is understood that all progeny may not be
identical to the parental cell since there may be mutations that occur
during replication. However, such progeny are included when the term "host
cell" is used. Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
In the present invention, the FHF-1 polynucleotide sequences may be
inserted into a recombinant expression vector. The term "recombinant
expression vector" refers to a plasmid, virus or other vehicle known in
the art that has been manipulated by insertion or incorporation of the
FHF-1 genetic sequences. Such expression vectors contain a promoter
sequence which facilitates the efficient transcription of the inserted
genetic sequence of the host. The expression vector typically contains an
origin of replication, a promoter, as well as specific genes which allow
phenotypic selection of the transformed cells. Vectors suitable for use in
the present invention include, but are not limited to the T7-based
expression vector for expression in bacteria (Rosenberg, et al., Gene,
56:125, 1987), the pMSXND expression vector for expression in mammalian
cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and
baculovirus-derived vectors for expression in insect cells. The DNA
segment can be present in the vector operably linked to regulatory
elements, for example, a promoter (e.g., T7, metallothionein I, or
polyhedrin promoters).
Polynucleotide sequences encoding FHF-1 can be expressed in either
prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and
mammalian organisms. Methods of expressing DNA sequences having eukaryotic
or viral sequences in prokaryotes are well known in the art. Biologically
functional vital and plasmid DNA vectors capable of expression and
replication in a host are known in the art. Such vectors are used to
incorporate DNA sequences of the invention.
Transformation of a host cell with recombinant DNA may be carried out by
conventional techniques as are well known to those skilled in the art.
Where the host is prokaryotic, such as E. coli, competent cells which are
capable of DNA uptake can be prepared from cells harvested after
exponential growth phase and subsequently treated by the CaCl.sub.2 method
using procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl
can be used. Transformation can also be performed after forming a
protoplast of the host cell if desired.
When the host is a eukaryote, such methods of transfection of DNA as
calcium phosphate co-precipitates, conventional mechanical procedures such
as microinjection, electroporation, insertion of a plasmid encased in
liposomes, or virus vectors may be used. Eukaryotic cells can also be
cotransformed with DNA sequences encoding the FHF-1 of the invention, and
a second foreign DNA molecule encoding a selectable phenotype, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic cells and
express the protein. (see for example, Eukaryotic Vital Vectors, Cold
Spring Harbor Laboratory, Gluzman ed., 1982).
Isolation and purification of microbial expressed polypeptide, or fragments
thereof, provided by the invention, may be carried out by conventional
means including preparative chromatography and immunological separations
involving monoclonal or polyclonal antibodies.
The FHF-1 polypeptides of the invention can also be used to produce
antibodies which are immunoreactive or bind to epitopes of the FHF-1
polypeptides. Antibody which consists essentially of pooled monoclonal
antibodies with different epitopic specificities, as well as distinct
monoclonal antibody preparations are provided. Monoclonal antibodies are
made from antigen containing fragments of the protein by methods well
known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols
in Molecular Biology, Ausubel, et al., ed., 1989).
The term "antibody" as used in this invention includes intact molecules as
well as fragments thereof, such as Fab, F(ab').sub.2, and Fv which are
capable of binding the epitopic determinant. These antibody fragments
retain some ability to selectively bind with its antigen or receptor and
are defined as follows:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment
of an antibody molecule can be produced by digestion of whole antibody
with the enzyme papain to yield an intact light chain and a portion of one
heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by treating
whole antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments are
obtained per antibody molecule;
(3) (Fab').sub.2, the fragment of the antibody that can be obtained by
treating whole antibody with the enzyme pepsin without subsequent
reduction; F(ab').sub.2 is a dimer of two Fab' fragments held together by
two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the
variable region of the light chain and the variable region of the heavy
chain expressed as two chains; and
(5) Single chain antibody ("SCA"), defined as a genetically engineered
molecule containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker as a
genetically fused single chain molecule.
Methods of making these fragments are known in the art. (See for example,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (1988), incorporated herein by reference).
As used in this invention, the term "epitope" means any antigenic
determinant on an antigen to which the paratope of an antibody binds.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three dimensional structural characteristics, as
well as specific charge characteristics.
Antibodies which bind to the FHF-1 polypeptide of the invention can be
prepared using an intact polypeptide or fragments containing small
peptides of interest as the immunizing antigen. The polypeptide or a
peptide used to immunize an animal can be derived from translated cDNA
(see for example, EXAMPLES 4 and 6) or chemical synthesis which can be
conjugated to a carrier protein, if desired. Such commonly used carriers
which are chemically coupled to the peptide include keyhole limpet
hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus
toxoid. The coupled peptide is then used to immunize the animal (e.g., a
mouse, a rat, or a rabbit).
If desired, polyclonal or monoclonal antibodies can be further purified,
for example, by binding to and elution from a matrix to which the
polypeptide or a peptide to which the antibodies were raised is bound.
Those of skill in the art will know of various techniques common in the
immunology arts for purification and/or concentration of polyclonal
antibodies, as well as monoclonal antibodies (See for example, Coligan, et
al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994,
incorporated by reference).
It is also possible to use the anti-idiotype technology to produce
monoclonal antibodies which mimic an epitope. For example, an
anti-idiotypic monoclonal antibody made to a first monoclonal antibody
will have a binding domain in the hypervariable region which is the
"image" of the epitope bound by the first monoclonal antibody.
The term "cell-proliferative disorder" denotes malignant as well as
non-malignant cell populations which often appear to differ from the
surrounding tissue both morphologically and genotypically. Malignant cells
(i.e. cancer) develop as a result of a multistep process. The FHF-1
polynucleotide that is an antisense molecule is useful in treating
malignancies of the various organ systems, particularly, for example,
cells in the central nervous system, including neural tissue, testes, and
cells of the eye. Essentially, any disorder which is etiologically linked
to altered expression of FHF-1 could be considered susceptible to
treatment with a FHF-1 suppressing reagent. One such disorder is a
malignant cell proliferative disorder, for example.
For purposes of the invention, an antibody or nucleic acid probe specific
for FHF-1 may be used to detect FHF-1 polypeptide (using antibody) or
polynucleotide (using nucleic acid probe) in biological fluids or tissues.
The invention provides a method for detecting a cell proliferative
disorder of neural tissue or testes, for example, which comprises
contacting an anti-FHF-1 antibody or nucleic acid probe with a cell
suspected of having a FHF-1 associated disorder and detecting binding to
the antibody or nucleic acid probe. The antibody reactive with FHF-1 or
the nucleic acid probe is preferably labeled with a compound which allows
detection of binding to FHF-1. Any specimen containing a detectable amount
of antigen or polynucleotide can be used. A preferred sample of this
invention is CNS, e.g., neural tissue or cerebrospinal fluid, testes, or
eye tissue. The level of FHF-1 in the suspect cell can be compared with
the level in a normal cell to determine whether the subject has a
FHF-1-associated cell proliferative disorder. Preferably the subject is
human.
When the cell component is nucleic acid, it may be necessary to amplify the
nucleic acid prior to binding with an FHF-1 specific probe. Preferably,
polymerase chain reaction (PCR) is used, however, other nucleic acid
amplification procedures such as ligase chain reaction (LCR), ligated
activated transcription (LAT) and nucleic acid sequence-based
amplification (NASBA) may be used.
The antibodies of the invention can be used in any subject in which it is
desirable to administer in vitro or in vivo immunodiagnosis or
immunotherapy. The antibodies of the invention are suited for use, for
example, in immunoassays in which they can be utilized in liquid phase or
bound to a solid phase carrier. In addition, the antibodies in these
immunoassays can be detectably labeled in various ways. Examples of types
of immunoassays which can utilize antibodies of the invention are
competitive and non-competitive immunoassays in either a direct or
indirect format. Examples of such immunoassays are the radioimmunoassay
(RIA) and the sandwich (immunometric) assay. Detection of the antigens
using the antibodies of the invention can be done utilizing immunoassays
which are run in either the forward, reverse, or simultaneous modes,
including immunohistochemical assays on physiological samples. Those of
skill in the art will know, or can readily discern, other immunoassay
formats without undue experimentation.
The antibodies of the invention can be bound to many different carriers and
used to detect the presence of an antigen comprising the polypeptide of
the invention. Examples of well-known carders include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and
modified celluloses, polyacrylamides, agaroses and magnetite. The nature
of the carrier can be either soluble or insoluble for purposes of the
invention. Those skilled in the art will know of other suitable carriers
for binding antibodies, or will be able to ascertain such, using routine
experimentation.
There are many different labels and methods of labeling known to those of
ordinary skill in the art. Examples of the types of labels which can be
used in the present invention include enzymes, radioisotopes, fluorescent
compounds, colloidal metals, chemiluminescent compounds, phosphorescent
compounds, and bioluminescent compounds. Those of ordinary skill in the
art will know of other suitable labels for binding to the antibody, or
will be able to ascertain such, using routine experimentation.
Another technique which may also result in greater sensitivity consists of
coupling the antibodies to low molecular weight haptens. These haptens can
then be specifically detected by means of a second reaction. For example,
it is common to use such haptens as biotin, which reacts with avidin, or
dinitrophenyl, puridoxal, and fluorescein, which can react with specific
antihapten antibodies.
In using the monoclonal antibodies of the invention for the in vivo
detection of antigen, the detectably labeled antibody is given a dose
which is diagnostically effective. The term "diagnostically effective"
means that the amount of detectably labeled monoclonal antibody is
administered in sufficient quantity to enable detection of the site having
the antigen comprising a polypeptide of the invention for which the
monoclonal antibodies are specific.
The concentration of detectably labeled monoclonal antibody which is
administered should be sufficient such that the binding to those cells
having the polypeptide is detectable compared to the background. Further,
it is desirable that the detectably labeled monoclonal antibody be rapidly
cleared from the circulatory system in order to give the best
target-to-background signal ratio.
As a rule, the dosage of detectably labeled monoclonal antibody for in vivo
diagnosis will vary depending on such factors as age, sex, and extent of
disease of the individual. Such dosages may vary, for example, depending
on whether multiple injections are given, antigenic burden, and other
factors known to those of skill in the art.
For in vivo diagnostic imaging, the type of detection instrument available
is a major factor in selecting a given radioisotope. The radioisotope
chosen must have a type of decay which is detectable for a given type of
instrument. Still another important factor in selecting a radioisotope for
in vivo diagnosis is that deleterious radiation with respect to the host
is minimized. Ideally, a radioisotope used for in vivo imaging will lack a
particle emission, but produce a large number of photons in the 140-250
keV range, which may readily be detected by conventional gamma cameras.
For in vivo diagnosis radioisotopes may be bound to immunoglobulin either
directly or indirectly by using an intermediate functional group.
Intermediate functional groups which often are used to bind radioisotopes
which exist as metallic ions to immunoglobulins are the bifunctional
chelating agents such as diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules. Typical
examples of metallic ions which can be bound to the monoclonal antibodies
of the invention are .sup.111 In, .sup.97 Ru, .sup.67 Ca, .sup.68 Ga,
.sup.72 As, .sup.89 Zr, and .sup.201 Tl.
The monoclonal antibodies of the invention can also be labeled with a
paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic
resonance imaging (MRI) or electron spin resonance (ESR). In general, any
conventional method for visualizing diagnostic imaging can be utilized.
Usually gamma and positron emitting radioisotopes are used for camera
imaging and paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157 Gd, .sup.55 Mn, .sup.162 Dy,
.sup.52 Cr, and .sup.56 Fe.
The monoclonal antibodies or polynucleotides of the invention can be used
in vitro and in vivo to monitor the course of amelioration of a
FHF-1-associated disease in a subject. Thus, for example, by measuring the
increase or decrease in the number of cells expressing antigen comprising
a polypeptide of the invention or changes in the concentration of such
antigen present in various body fluids, it would be possible to determine
whether a particular therapeutic regimen aimed at ameliorating the
FHF-1-associated disease is effective. The term "ameliorate" denotes a
lessening of the detrimental effect of the FHF-1-associated disease in the
subject receiving therapy.
The present invention identifies a nucleotide sequence that can be
expressed in an altered manner as compared to expression in a normal cell,
therefore it is possible to design appropriate therapeutic or diagnostic
techniques directed to this sequence. Detection of elevated levels of
FHF-1 expression is accomplished by hybridization of nucleic acids
isolated from a cell suspected of having an FHF-1 associated proliferative
disorder with an FHF-1 polynucleotide of the invention. Analysis, such as
Northern Blot analysis, are utilized to quantitate expression of FHF-1.
Other standard nucleic acid detection techniques will be known to those of
skill in the art.
Treatment of an FHF-1 associated cell proliferative disorder include
modulation of FI-IF-1 gene expression and FHF-1 activity. The term
"modulate" envisions the suppression of expression of FHF-1 when it is
over-expressed, or augmentation of FHF-1 expression when it is
under-expressed. Where a cell-proliferative disorder is associated with
the expression of FHF-1, nucleic acid sequences that interfere with FHF-1
expression at the translational level can be used. This approach utilizes,
for example, antisense nucleic acid, ribozymes, or triplex agents to block
transcription or translation of a specific FHF-1 mRNA, either by masking
that mRNA with an antisense nucleic acid or triplex agent, or by cleaving
it with a ribozyme. Such disorders include neurodegenerative diseases, for
example.
Antisense nucleic acids are DNA or RNA molecules that are complementary to
at least a portion of a specific mRNA molecule (Weintraub, Scientific
American, 262:40, 1990). In the cell, the antisense nucleic acids
hybridize to the corresponding mRNA, forming a double-stranded molecule.
The antisense nucleic acids interfere with the translation of the mRNA,
since the cell will not translate a mRNA that is double-stranded.
Antisense oligomers of about 15 nucleotides are preferred, since they are
easily synthesized and are less likely to cause problems than larger
molecules when introduced into the target FHF-1-producing cell. The use of
antisense methods to inhibit the in vitro translation of genes is well
known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).
Use of an oligonucleotide to stall transcription is known as the triplex
strategy since the oligomer winds around double-helical DNA, forming a
three-strand helix. Therefore, these triplex compounds can be designed to
recognize a unique site on a chosen gene (Maher, et al., Antisense Res.
and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design, 6(.6):569,
1991).
Ribozymes are RNA molecules possessing the ability to specifically cleave
other single-stranded RNA in a manner analogous to DNA restriction
endonucleases. Through the modification of nucleotide sequences which
encode these RNAs, it is possible to engineer molecules that recognize
specific nucleotide sequences in an RNA molecule and cleave it (Cech, J.
Amer. Med Assn., 260:3030, 1988). A major advantage of this approach is
that, because they are sequence-specific, only mRNAs with particular
sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type
(Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.
Tetrahymena-type ribozymes recognize sequences which are four bases in
length, while "hammerhead"-type ribozymes recognize base sequences 11-18
bases in length. The longer the recognition sequence, the greater the
likelihood that the sequence will occur exclusively in the target mRNA
species. Consequently, hammerhead-type ribozymes are preferable to
tetrahymena-type ribozymes for inactivating a specific mRNA species and
18-based recognition sequences are preferable to shorter recognition
sequences.
The present invention also provides gene therapy for the treatment of cell
proliferative or immunologic disorders which are mediated by FHF-1
protein. Such therapy would achieve its therapeutic effect by introduction
of the FHF-1 antisense polynucleotide into cells having the proliferative
disorder. Delivery of antisense FHF-1 polynucleotide can be achieved using
a recombinant expression vector such as a chimeric virus or a colloidal
dispersion system. Especially preferred for therapeutic delivery of
antisense sequences is the use of targeted liposomes.
Various viral vectors which can be utilized for gene therapy as taught
herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA
virus such as a retrovirus. Preferably, the retrovital vector is a
derivative of a murine or avian retrovirus. Examples of retroviral vectors
in which a single foreign gene can be inserted include, but are not
limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma
virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus
(RSV). Preferably, when the subject is a human, a vector such as the
gibbon ape leukemia virus (GaLV) is utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these vectors
can transfer or incorporate a gene for a selectable marker so that
transduced cells can be identified and generated. By inserting a FHF-1
sequence of interest into the viral vector, along with another gene which
encodes the ligand for a receptor on a specific target cell, for example,
the vector is now target specific. Retrovital vectors can be made target
specific by attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target the
retroviral vector. Those of skill in the art will know of, or can readily
ascertain without undue experimentation, specific polynucleotide sequences
which can be inserted into the retrovital genome or attached to a vital
envelope to allow target specific delivery of the retroviral vector
containing the FHF-1 antisense polynucleotide.
Since recombinant retroviruses are defective, they require assistance in
order to produce infectious vector particles. This assistance can be
provided, for example, by using helper cell lines that contain plasmids
encoding all of the structural genes of the retrovirus under the control
of regulatory sequences within the LTR. These plasmids are missing a
nucleotide sequence which enables the packaging mechanism to recognize an
RNA transcript for encapsidation. Helper cell lines which have deletions
of the packaging signal include, but are not limited to .PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no genome
is packaged. If a retroviral vector is introduced into such cells in which
the packaging signal is intact, but the structural genes are replaced by
other genes of interest, the vector can be packaged and vector virion
produced.
Alternatively, NIH 3T3 or other tissue culture cells can be directly
transfected with plasmids encoding the retroviral structural genes gag,
pol and env, by conventional calcium phosphate transfection. These cells
are then transfected with the vector plasmid containing the genes of
interest. The resulting cells release the retroviral vector into the
culture medium.
Another targeted delivery system for FHF-1 antisense polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles, mixed
micelles, and liposomes. The preferred colloidal system of this invention
is a liposome. Liposomes are artificial membrane vesicles which are useful
as delivery vehicles in vitro and in vivo. It has been shown that large
unilamellar vesicles (LUV), which range in size from 0.2-4.0 .mu.m can
encapsulate a substantial percentage of an aqueous buffer containing large
macromolecules. RNA, DNA and intact virions can be encapsulated within the
aqueous interior and be delivered to cells in a biologically active form
(Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to
mammalian cells, liposomes have been used for delivery of polynucleotides
in plant, yeast and bacterial cells. In order for a liposome to be an
efficient gene transfer vehicle, the following characteristics should be
present: (1) encapsulation of the genes of interest at high efficiency
while not compromising their biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target cells;
(3) delivery of the aqueous contents of the vesicle to the target cell
cytoplasm at high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
The composition of the liposome is usually a combination of phospholipids,
particularly high-phase-transition-temperature phospholipids, usually in
combination with steroids, especially cholesterol. Other phospholipids or
other lipids may also be used. The physical characteristics of liposomes
depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl
compounds, such as phosphatidylglycerol, phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides,
and gangliosides. Particularly useful are diacylphosphatidylglycerols,
where the lipid moiety contains from 14-18 carbon atoms, particularly from
16-18 carbon atoms, and is saturated. Illustrative phospholipids include
egg phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
The targeting of liposomes can be classified based on anatomical and
mechanistic factors. Anatomical classification is based on the level of
selectivity, for example, organ-specific, cell-specific, and
organelle-specific. Mechanistic targeting can be distinguished based upon
whether it is passive or active. Passive targeting utilizes the natural
tendency of liposomes to distribute to cells of the reticulo-endothelial
system (RES) in organs which contain sinusoidal capillaries. Active
targeting, on the other hand, involves alteration of the liposome by
coupling the liposome to a specific ligand such as a monoclonal antibody,
sugar, glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types other
than the naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of
ways. In the case of a liposomal targeted delivery system, lipid groups
can be incorporated into the lipid bilayer of the liposome in order to
maintain the targeting ligand in stable association with the liposomal
bilayer. Various linking groups can be used for joining the lipid chains
to the targeting ligand.
Due to the expression of FHF-1 in the testes, eye and brain, or neural
tissue, there are a variety of applications using the polypeptide,
polynucleotide, and antibodies of the invention, related to these tissues.
Such applications include treatment of cell proliferative and immunologic
disorders involving these and other tissues. In addition, FHF-1 may be
useful in various gene therapy procedures.
Due to the high level of expression of FHF-1 in the testes, there are a
variety of applications using the polypeptide, polynucleotide, and
antibodies of the invention related to this tissue. Such applications
include treatment of cell proliferative disorders associated with FHF-1
expression in the testes. Various testicular developmental or acquired
disorders can also be subject to FHF-1 applications. These may include,
but are not limited to vital infection (e.g., vital orchitis),
autoimmunity, sperm production or dysfunction, trauma, and testicular
tumors. The presence of high levels of FHF-1 in the testis suggests that
FHF-1 or an analogue of FHF-1 could be used to increase or decrease male
fertility.
The identification of a novel member of the FGF family provides a useful
tool for diagnosis, prognosis and therapeutic strategies associated with
FHF-1 mediated disorders. Measurement of FHF-1 levels using anti-FHF-1
antibodies is a useful diagnostic for following the progression or
recovery from diseases of the nervous system, including: cancer, stroke,
neurodegenerative diseases such as Parkinson's disease or Alzheimer's
disease, retinal diseases such as retinitis pigmentosa, or viral
encephalitis. The presence of high levels of FHF-1 in the central nervous
system suggests that the observed low level of FHF-1 in a number of
peripheral tissues could reflect FHF-1 in peripheral nerve, and therefore
measurement of FHF-1 levels using anti-FHF-1 antibodies could be
diagnostic for peripheral neuropathy. The presence of high levels of FHF-1
in the testis suggests that measurement of FHF-1 levels using anti-FHF-1
antibodies could be diagnostic for testicular cancer.
Like other members of the FGF family, FHF-1 likely has mitogenic and/or
cell survival activity, therefore FHF-1 or an analogue that mimics FHF-1
action could be used to promote tissue repair or replacement. The presence
of FHF-1 in the CNS suggests such a therapeutic role in diseases of the
nervous system, including: stroke, neurodegenerative diseases such as
Parkinson's disease or Alzheimer's disease, or in retinal degenerative
diseases such as retinitis pigmentosa or macular degeneration, or in
peripheral neuropathies. Conversely, blocking FHF-1 action either with
anti-FHF-1 antibodies or with an FHF-1 antagonist might slow or ameliorate
diseases in which excess cell growth is pathological, most obviously
cancer.
The following examples are intended to illustrate but not limit the
invention. While they are typical of those that might be used, other
procedures known to those skilled in the art may alternatively be used.
EXAMPLE 1
IDENTIFICATION OF FHF-1, A NOVEL MEMBER OF THE FGF FAMILY
To identify novel gene products expressed in the human retina, random
segments of human retina cDNA clones were partially sequenced, and the
resulting partial sequences compared to the sequences available in the
public databases.
In detail, an adult human retina cDNA library constructed in lambda gt10
(Nathans, et al., Science 232:193, 1986) was amplified, and the cDNA
inserts were excised en mass by cleavage with EcoR I and purified free of
the vector by agarose gel electrophoresis. Following heat denaturation of
the purified cDNA inserts, a synthetic oligonucleotide containing an EcoR
I site at its 5' end and six random nucleotides at its 3' end (5'
GACGAGATATTAGAATTCTACTCGNNNNNN) (SEQ ID NO :3) was used to prime two
sequential rounds of DNA synthesis in the presence of the Klenow fragment
of E. coli DNA polymerase. The resulting duplex molecules were amplified
using the polymerase chain reaction (PCR) with a primer corresponding to
the unique 5' flanking sequence (5' CCCCCCCCCGACGAGATATTAGAATTCTACTCG)
(SEQ ID NO:4). These PCR products, representing a random sampling of the
original cDNA inserts, were cleaved with EcoR I, size fractionated by
preparative agarose gel electrophoresis to include only segments of
approximately 500 bp in length, and cloned into lambda gt10. Three
thousand single plaques from this derivative library were arrayed in
96-well trays and from these clones the inserts were amplified by PCR
using flanking vector primers and then sequenced using the dideoxy method
and automated fluorescent detection (Applied Biosystems). A single
sequencing run from one end of each insert was conceptually translated on
both strands in all three reading frames and the six resulting amino acid
sequences were used to search for homology in the GenBank nonredundant
protein database using the BLASTX searching algorithm.
One partial cDNA sequence was found that showed statistically significant
homology to previously described members of the FGF family. Using this
partial cDNA as a probe, multiple independent cDNA clones were isolated
from the human retina cDNA library, including two that encompass the
entire open reading frame and from which complete nucleotide sequences
were determined.
EXAMPLE 2
DEDUCED PRIMARY STRUCTURE OF FHF-1
FIG. 1 shows the sequence of human FHF-1 deduced from the nucleotide
sequences of two independent human retina cDNA clones. The primary
translation product of human FHF-1 is predicted to be 244 amino acids in
length. The human FHF-1 inititiator methionine codon shown in FIG. 1 at
position 332-334 is the first ATG codon following the in-frame stop codon
at nucleotides 323-325; a good consensus ribosome binding site (CAGCTATGG
(SEQ ID NO:5); Kozak, Nucleic Acids Res. 15: 8125, 1987) is found at this
position. The next methionine codon within the open reading frame is
encountered 86 codons 3' of the putative initiator methionine codon. As
observed for aFGF and bFGF, the amino-terminus of the primary translation
product of FHF-1 does not conform to the consensus sequence for a signal
peptide to direct cotranslational insertion across the endoplasmic
reticulum membrane. The FHF-1 sequence lacks asn-X-ser/thr sites for
asparagine-linked glycosylation.
Alignment of FHF-1 with the other known members of the FGF family is shown
in FIG. 2 and a dendrogram showing the degree of amino acid similarity is
shown in FIG. 3. The most homologous FGF family member is FGF-9 which
shows 27% amino acid identity with FHF-1 when aligned with 10 gaps. Note
that in the central region of each polypeptide, all FGF family members,
including FHF-1, share 11 invariant amino acids.
EXAMPLE 3
CHROMOSOMAL LOCALIZATION OF FHF-1
The chromosomal location of FHF-1 was determined by probing a Southern blot
containing restriction enzyme digested DNA derived from a panel of 24
human-mouse and human-hamster cell lines, each containing a different
human chromosome (Oncor, Gaithersburg, Md.). As seen in FIG. 4,
hybridization of the human FHF-1 probe to human, mouse, and hamster
genomic DNA produces distinct hybridizing fragment sizes. Among the hybrid
panels, the human-specific hybridization pattern is seen only in the lane
corresponding to the hybrid cell line carrying human chromosome 3.
EXAMPLE 4
PRODUCTION OF FHF-1 IN TRANSFECTED HUMAN CELLS
To express FHF-1 in human cells, the complete open reading frame was
inserted into the eukaryotic expression vector pCIS (Gorman, et al., DNA
Protein Eng. Tech. 2: 3, 1990). To increase the efficiency of translation,
the region immediately 5' of the initiator methionine coding was converted
to an optimal ribosome binding site (CCACCATGG) by PCR amplification with
a primer that carded the desired sequence. Following transient
transfection of human embryonic kidney cells with the expression construct
and a plasmid expressing the simian virus 40 (SV40) large T-antigen
(pRSV-TAg; Gorman, et al., supra), cells were metabolically labeled with
.sup.32 S methionine for 6 hours in the absence of serum. As shown in FIG.
5, cells transfected with FHF-1 synthesize a single abundant polypeptide
with an apparent molecular mass of 30 kD that is not produced by cells
transfected with either of two unrelated constructs. This polypeptide
corresponds closely to the predicted molecular mass of the primary
translation product, 27.4 kD. FIG. 5 also shows that cells transfected
with a human growth hormone (hGH) expression plasmid efficiently secrete
hGH, whereas FHF-1 accumulates within the transfected cells and fails to
be secreted in detectable quantities.
EXAMPLE 5
TISSUE DISTRIBUTION OF FHF-1 mRNA
To determine the tissue distribution of FHF-1 mRNA, RNase protection
analysis was performed on total RNA from mouse brain, eye, heart, kidney,
liver, lung, spleen, and testis, as well as a yeast tRNA negative control.
The probe used was derived from a segment of the mouse FHF-1 gene isolated
by hybridization with the full-length human FHF-1 cDNA. As seen in FIG. 6,
the highest levels of FHF-1 expression are in the brain, eye, and testis.
Very low levels of FHF-1 expression were detected in kidney, liver, and
lung on a five-fold longer exposure of the autoradiogram.
EXAMPLE 6
PRODUCTION OF ANTIBODIES SPECIFIC FOR FHF-1
To generate anti-FI-IF-1 antibodies, a DNA segment encompassing the
carboxy-terminal 190 amino acids of FHF-1 was inserted into the E. coli
expression vector pGEMEX (Studier, et al, Meth. Enzymol. 185: 60, 1990).
The recombinant fusion protein between the T7 gene 10 protein and the
carboxy-terminal 190 amino acids of FHF-1 was produced in E. coli,
purified by preparative polyacrylamide gel electrophoresis, and used to
immunize rabbits. Anti-FI-IF-1 antibodies from immune serum were affinity
purified using the fusion protein immobilized onto nitrocellulose; those
antibodies directed against the pGEMEX T7 gene10 protein fusion partner
were removed by absorption to the purified T7 gene 10 protein immobilized
onto nitrocellulose. By Western blotting, the affinity purified anti-FHF-1
antibodies were shown to recognize recombinant FHF-1 produced either in E.
coli or in human embryonic kidney cells. By immunohistochemical staining
the antibodies also specifically recognized recombinant FHF-1 produced in
human embryonic kidney cells transfected with the FHF-1 expression plasmid
described above. Immuno-staining of neural tissues shows anti-FHF-1
immunostaining in the ganglion cell layer and inner nuclear layers of
adult mouse and macaque monkey retinas and in a large number of regions
within the adult mouse brain.
Although the invention has been described with reference to the presently
preferred embodiment, it should be understood that various modifications
can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 15
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1422 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 332..1060
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GAATTCCGCACACTGCGTTCGGGGTACCAAGTGGAAGGGGAAGAACGATGCCCAAAATAA60
CAAGACGTGCCTGGGACCGCCCCGCCCCGCCCCCCGGCCGCCAGAGGTTGGGGAAGTTTA120
CATCTGGATTTTCACACATTTTGTCGCCACTGCCCAGACTTTGACTAACCTTGTGAGCGC180
CGGGTTTTCGATACTGCAGCCTCCTCAAATTTTAGCACTGCCTCCCCGCGACTGCCCTTT240
CCCTGGCCGCCCAGGTCCTGCCCTCGCCCCGGCGGAGCGCAAGCCGGAGGGCGCAGTAGA300
GGCTGGGGCCTGAGGCCCTCGCTGAGCAGCTATGGCTGCGGCGATAGCCAGC352
MetAlaAlaAlaIleAlaSer
15
TCCTTGATCCGGCAGAAGCGGCAGGCGAGGGAGTCCAACAGCGACCGA400
SerLeuIleArgGlnLysArgGlnAlaArgGluSerAsnSerAspArg
101520
GTGTCGGCCTCCAAGCGCCGCTCCAGCCCCAGCAAAGACGGGCGCTCC448
ValSerAlaSerLysArgArgSerSerProSerLysAspGlyArgSer
253035
CTGTGCGAGAGGCACGTCCTCGGGGTGTTCAGCAAAGTGCGCTTCTGC496
LeuCysGluArgHisValLeuGlyValPheSerLysValArgPheCys
40455055
AGCGGCCGCAAGAGGCCGGTGAGGCGGAGACCAGAACCCCAGCTCAAA544
SerGlyArgLysArgProValArgArgArgProGluProGlnLeuLys
606570
GGGATTGTGACAAGGTTATTCAGCCAGCAGGGATACTTCCTGCAGATG592
GlyIleValThrArgLeuPheSerGlnGlnGlyTyrPheLeuGlnMet
758085
CACCCAGATGGTACCATTGATGGGACCAAGGACGAAAACAGCGACTAC640
HisProAspGlyThrIleAspGlyThrLysAspGluAsnSerAspTyr
9095100
ACTCTCTTCAATCTAATTCCCGTGGGCCTGCGTGTAGTGGCCATCCAA688
ThrLeuPheAsnLeuIleProValGlyLeuArgValValAlaIleGln
105110115
GGAGTGAAGGCTAGCCTCTATGTGGCCATGAATGGTGAAGGCTATCTC736
GlyValLysAlaSerLeuTyrValAlaMetAsnGlyGluGlyTyrLeu
120125130135
TACAGTTCAGATGTTTTCACTCCAGAATGCAAATTCAAGGAATCTGTG784
TyrSerSerAspValPheThrProGluCysLysPheLysGluSerVal
140145150
TTTGAAAACTACTATGTGATCTATTCTTCCACACTGTACCGCCAGCAA832
PheGluAsnTyrTyrValIleTyrSerSerThrLeuTyrArgGlnGln
155160165
GAATCAGGCCGAGCTTGGTTTCTGGGACTCAATAAAGAAGGTCAAATT880
GluSerGlyArgAlaTrpPheLeuGlyLeuAsnLysGluGlyGlnIle
170175180
ATGAAGGGGAACAGAGTGAAGAAAACCAAGCCCTCATCACATTTTGTA928
MetLysGlyAsnArgValLysLysThrLysProSerSerHisPheVal
185190195
CCGAAACCTATTGAAGTGTGTATGTACAGAGAACCATCGCTACATGAA976
ProLysProIleGluValCysMetTyrArgGluProSerLeuHisGlu
200205210215
ATTGGAGAAAAACAAGGGCGTTCAAGGAAAAGTTCTGGAACACCAACC1024
IleGlyGluLysGlnGlyArgSerArgLysSerSerGlyThrProThr
220225230
ATGAATGGAGGCAAAGTTGTGAATCAAGATTCAACATAGCTGAGAA1070
MetAsnGlyGlyLysValValAsnGlnAspSerThr
235240
CTCTCCCCTTCTTCCCTCTCTCATCCCTTCCCCTTCCCTTCCTTCCCATTTACCCATTTC1130
CTTCCAGTAAATCCACCCAAGGAGAGGAAAATAAAATGACAACGCAAGACCTAGTGGCTA1190
AGATTCTGCACTCAAAATCTTCCTTTGTGTAGGACAAGAAAATTGAACCAAAGCTTGCTT1250
GTTGCAATGTGGTAGAAAATTCACGTGCACAAAGATTAGCACACTTAAAAGCAAAGGAAA1310
AAATAAATCAGAACTCCATAAATATTAAACTAAACTGTATTGTTATTAGTAGAAGGCTAA1370
TTGTAATGAAGACATTAATAAAGATGAAATAAACTTATTACTTTCGGAATTC1422
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetAlaAlaAlaIleAlaSerSerLeuIleArgGlnLysArgGlnAla
151015
ArgGluSerAsnSerAspArgValSerAlaSerLysArgArgSerSer
202530
ProSerLysAspGlyArgSerLeuCysGluArgHisValLeuGlyVal
354045
PheSerLysValArgPheCysSerGlyArgLysArgProValArgArg
505560
ArgProGluProGlnLeuLysGlyIleValThrArgLeuPheSerGln
65707580
GlnGlyTyrPheLeuGlnMetHisProAspGlyThrIleAspGlyThr
859095
LysAspGluAsnSerAspTyrThrLeuPheAsnLeuIleProValGly
100105110
LeuArgValValAlaIleGlnGlyValLysAlaSerLeuTyrValAla
115120125
MetAsnGlyGluGlyTyrLeuTyrSerSerAspValPheThrProGlu
130135140
CysLysPheLysGluSerValPheGluAsnTyrTyrValIleTyrSer
145150155160
SerThrLeuTyrArgGlnGlnGluSerGlyArgAlaTrpPheLeuGly
165170175
LeuAsnLysGluGlyGlnIleMetLysGlyAsnArgValLysLysThr
180185190
LysProSerSerHisPheValProLysProIleGluValCysMetTyr
195200205
ArgGluProSerLeuHisGluIleGlyGluLysGlnGlyArgSerArg
210215220
LysSerSerGlyThrProThrMetAsnGlyGlyLysValValAsnGln
225230235240
AspSerThr
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GACGAGATATTAGAATTCTACTCGNNNNNN30
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCCCCCCCCGACGAGATATTAGAATTCTACTCG33
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CAGCTATGG9
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetGlySerProArgSerAlaLeuSerCysLeuLeuLeuHisLeuLeu
151015
ValLeuCysLeuGlnAlaGlnValThrValGlnSerSerProAsnPhe
202530
ThrGlnHisValArgGluGlnSerLeuValThrAspGlnLeuSerArg
354045
ArgLeuIleArgThrTyrGlnLeuTyrSerArgThrSerGlyLysHis
505560
ValGlnValLeuAlaAsnLysArgIleAsnAlaMetAlaGluAspGly
65707580
AspProPheAlaLysLeuIleValGluThrAspThrPheGlySerArg
859095
ValArgValArgGlyAlaGluThrGlyLeuTyrIleCysMetAsnLys
100105110
LysGlyLysLeuIleAlaLysSerAsnGlyLysGlyLysAspCysVal
115120125
PheIleGluIleValLeuGluAsnAsnTyrThrAlaLeuGlnAsnAla
130135140
LysTyrGluGlyTrpTyrMetAlaPheThrArgLysGlyArgProArg
145150155160
LysGlySerLysThrArgGlnHisGlnArgGluValHisPheMetLys
165170175
ArgLeuProArgGlyHisHisThrThrGluGlnSerLeuArgPheGlu
180185190
PheLeuAsnTyrProProPheThrArgSerLeuArgGlySerGlnArg
195200205
ThrTrpAlaProGluProArg
210215
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
MetAlaProLeuGlyGluValGlyAsnTyrPheGlyValGlnAspAla
151015
ValProPheGlyAsnValProValLeuProValAspSerProValLeu
202530
LeuSerAspHisLeuGlyGlnSerGluAlaGlyGlyLeuProArgGly
354045
ProAlaValThrAspLeuAspHisLeuLysGlyIleLeuArgArgArg
505560
GlnLeuTyrCysArgThrGlyPheHisLeuGluIlePheProAsnGly
65707580
ThrIleGlnGlyThrArgLysAspHisSerArgPheGlyIleLeuGlu
859095
PheIleSerIleAlaValGlyLeuValSerIleArgGlyValAspSer
100105110
GlyLeuTyrLeuGlyMetAsnGluLysGlyGluLeuTyrGlySerGlu
115120125
LysLeuThrGlnGluCysValPheArgGluGlnPheGluGluAsnTrp
130135140
TyrAsnThrTyrSerSerAsnLeuTyrLysHisValAspThrGlyArg
145150155160
ArgTyrTyrValAlaLeuAsnLysAspGlyThrProArgGluGlyThr
165170175
ArgThrLysArgHisGlnLysPheThrHisPheLeuProArgProVal
180185190
AspProAspLysValProGluLeuTyrLysAspIleLeuSerGlnSer
195200205
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetAlaAlaAlaIleAlaSerSerLeuIleArgGlnLysArgGlnAla
151015
ArgGluSerAsnSerAspArgValSerAlaSerLysArgArgSerSer
202530
ProSerLysAspGlyArgSerLeuCysGluArgHisValLeuGlyVal
354045
PheSerLysValArgPheCysSerGlyArgLysArgProValArgArg
505560
ArgProGluProGlnLeuLysGlyIleValThrArgLeuPheSerGln
65707580
GlnGlyTyrPheLeuGlnMetHisProAspGlyThrIleAspGlyThr
859095
LysAspGluAsnSerAspTyrThrLeuPheAsnLeuIleProValGly
100105110
LeuArgValValAlaIleGlnGlyValLysAlaSerLeuTyrValAla
115120125
MetAsnGlyGluGlyTyrLeuTyrSerSerAspValPheThrProGlu
130135140
CysLysPheLysGluSerValPheGluAsnTyrTyrValIleTyrSer
145150155160
SerThrLeuTyrArgGlnGlnGluSerGlyArgAlaTrpPheLeuGly
165170175
LeuAsnLysGluGlyGlnIleMetLysGlyAsnArgValLysLysThr
180185190
LysProSerSerHisPheValProLysProIleGluValCysMetTyr
195200205
ArgGluProSerLeuHisGluIleGlyGluLysGlnGlyArgSerArg
210215220
LysSerSerGlyThrProThrMetAsnGlyGlyLysValValAsnGln
225230235240
AspSerThr
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
MetAlaGluGlyGluIleThrThrPheThrAlaLeuThrGluLysPhe
151015
AsnLeuProProGlyAsnTyrLysLysProLysLeuLeuTyrCysSer
202530
AsnGlyGlyHisPheLeuArgIleLeuProAspGlyThrValAspGly
354045
ThrArgAspArgSerAspGlnHisIleGlnLeuGlnLeuSerAlaGlu
505560
SerValGlyGluValTyrIleLysSerThrGluThrGlyGlnTyrLeu
65707580
AlaMetAspThrAspGlyLeuLeuTyrGlySerGlnThrProAsnGlu
859095
GluCysLeuPheLeuGluArgLeuGluGluAsnHisTyrAsnThrTyr
100105110
IleSerLysLysHisAlaGluLysAsnTrpPheValGlyLeuLysLys
115120125
AsnGlySerCysLysArgGlyProArgThrHisTyrGlyGlnLysAla
130135140
IleLeuPheLeuProLeuProValSerSerAsp
145150155
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
MetAlaAlaGlySerIleThrThrLeuProAlaLeuProGluAspGly
151015
GlySerGlyAlaPheProProGlyHisPheLysAspProLysArgLeu
202530
TyrCysLysAsnGlyGlyPhePheLeuArgIleHisProAspGlyArg
354045
ValAspGlyValArgGluLysSerAspProHisIleLysLeuGlnLeu
505560
GlnAlaGluGluArgGlyValValSerIleLysGlyValCysAlaAsn
65707580
ArgTyrLeuAlaMetLysGluAspGlyArgLeuLeuAlaSerLysCys
859095
ValThrAspGluCysPhePhePheGluArgLeuGluSerAsnAsnTyr
100105110
AsnThrTyrArgSerArgLysTyrThrSerTrpTyrValAlaLeuLys
115120125
ArgThrGlyGlnTyrLysLeuGlySerLysThrGlyProGlyGlnLys
130135140
AlaIleLeuPheLeuProMetSerAlaLysSer
145150155
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
MetGlyLeuIleTrpLeuLeuLeuLeuSerLeuLeuGluProSerTrp
151015
ProThrThrGlyProGlyThrArgLeuArgArgAspAlaGlyGlyArg
202530
GlyGlyValTyrGluHisLeuGlyGlyAlaProArgArgArgLysLeu
354045
TyrCysAlaThrLysTyrHisLeuGlnLeuHisProSerGlyArgVal
505560
AsnGlySerLeuGluAsnSerAlaTyrSerIleLeuGluIleThrAla
65707580
ValGluValGlyValValAlaIleLysGlyLeuPheSerGlyArgTyr
859095
LeuAlaMetAsnLysArgGlyArgLeuTyrAlaSerAspHisTyrAsn
100105110
AlaGluCysGluPheValGluArgIleHisGluLeuGlyTyrAsnThr
115120125
TyrAlaSerArgLeuTyrArgThrGlySerSerGlyProGlyAlaGln
130135140
ArgGlnProGlyAlaGlnArgProTrpTyrValSerValAsnGlyLys
145150155160
GlyArgProArgArgGlyPheLysThrArgArgThrGlnLysSerSer
165170175
LeuPheLeuProArgValLeuGlyHisLysAspHisGluMetValArg
180185190
LeuLeuGlnSerSerGlnProArgAlaProGlyGluGlySerGlnPro
195200205
ArgGlnArgArgGlnLysLysGlnSerProGlyAspHisGlyLysMet
210215220
GluThrLeuSerThrArgAlaThrProSerThrGlnLeuHisThrGly
225230235240
GlyLeuAlaValAla
245
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
MetSerLeuSerPheLeuLeuLeuLeuPhePheSerHisLeuIleLeu
151015
SerAlaTrpAlaHisGlyGluLysArgLeuAlaProLysGlyGlnPro
202530
GlyProAlaAlaThrAspArgAsnProIleGlySerSerSerArgGln
354045
SerSerSerSerAlaMetSerSerSerSerAlaSerSerSerProAla
505560
AlaSerLeuGlySerGlnGlySerGlyLeuGluGlnSerSerPheGln
65707580
TrpSerProSerGlyArgArgThrGlySerLeuTyrCysArgValGly
859095
IleGlyPheHisLeuGlnIleTyrProAspGlyLysValAsnGlySer
100105110
HisGluAlaAsnMetLeuSerValLeuGluIlePheAlaValSerGln
115120125
GlyIleValGlyIleArgGlyValPheSerAsnLysPheLeuAlaMet
130135140
SerLysLysGlyLysLeuHisAlaSerAlaLysPheThrAspAspCys
145150155160
LysPheArgGluArgPheGlnGluAsnSerTyrAsnThrTyrAlaSer
165170175
AlaIleHisArgThrGluLysThrGlyArgGluTrpTyrValAlaLeu
180185190
AsnLysArgGlyLysAlaLysArgGlyCysSerProArgValLysPro
195200205
GlnHisIleSerThrHisPheLeuProArgPheLysGlnSerGluGln
210215220
ProGluLeuSerPheThrValThrValProGluLysLysAsnProPro
225230235240
SerProIleLysSerLysIleProLeuSerAlaProArgLysAsnThr
245250255
AsnSerValLysTyrArgLeuLysPheArgPheGly
260265
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
MetSerGlyProGlyThrAlaAlaValAlaLeuLeuProAlaValLeu
151015
LeuAlaLeuLeuAlaProTrpAlaGlyArgGlyGlyAlaAlaAlaPro
202530
ThrAlaProAsnGlyThrLeuGluAlaGluLeuGluArgArgTrpGlu
354045
SerLeuValAlaLeuSerLeuAlaArgLeuProValAlaAlaGlnPro
505560
LysGluAlaAlaValGlnSerGlyAlaGlyAspTyrLeuLeuGlyIle
65707580
LysArgLeuArgArgLeuTyrCysAsnValGlyIleGlyPheHisLeu
859095
GlnAlaLeuProAspGlyArgIleGlyGlyAlaHisAlaAspThrArg
100105110
AspSerLeuLeuGluLeuSerProValGluArgGlyValValSerIle
115120125
PheGlyValAlaSerArgPhePheValAlaMetSerSerLysGlyLys
130135140
LeuTyrGlySerProPhePheThrAspGluCysIlePheLysGluIle
145150155160
LeuLeuProAsnAsnTyrAsnAlaTyrGluSerTyrLysTyrProGly
165170175
MetPheIleAlaLeuSerLysAsnGlyLysThrLysLysGlyAsnArg
180185190
ValSerProThrMetLysValThrHisPheLeuProArgLeu
195200205
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
MetSerArgGlyAlaGlyArgLeuGlnGlyThrLeuTrpAlaLeuVal
151015
PheLeuGlyIleLeuValGlyMetValValProSerProAlaGlyThr
202530
ArgAlaAsnAsnThrLeuLeuAspSerArgGlyTrpGlyThrLeuLeu
354045
SerArgSerArgAlaGlyLeuAlaGlyGluIleAlaGlyValAsnTrp
505560
GluSerGlyTyrLeuValGlyIleLysArgGlnArgArgLeuTyrCys
65707580
AsnValGlyIleGlyPheHisLeuGlnValLeuProAspGlyArgIle
859095
SerGlyThrHisGluGluAsnProTyrSerLeuLeuGluIleSerThr
100105110
ValGluArgGlyValValSerLeuPheGlyValArgSerAlaLeuPhe
115120125
ValAlaMetAsnSerLysGlyArgLeuTyrAlaThrProSerPheGln
130135140
GluGluCysLysPheArgGluThrLeuLeuProAsnAsnTyrAsnAla
145150155160
TyrGluSerAspLeuTyrGlnGlyThrTyrIleAlaLeuSerLysTyr
165170175
GlyArgValLysArgGlySerLysValSerProIleMetThrValThr
180185190
HisPheLeuProArgIle
195
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Not Relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
MetHisLysTrpIleLeuThrTrpIleLeuProThrLeuLeuTyrArg
151015
SerCysPheHisIleIleCysLeuValGlyThrIleSerLeuAlaCys
202530
AsnAspMetThrProGluGlnMetAlaThrAsnValAsnCysSerSer
354045
ProGluArgHisThrArgSerTyrAspTyrMetGluGlyGlyAspIle
505560
ArgValArgArgLeuPheCysArgThrGlnTrpTyrLeuArgIleAsp
65707580
LysArgGlyLysValLysGlyThrGlnGluMetLysAsnAsnTyrAsn
859095
IleMetGluIleArgThrValAlaValGlyIleValAlaIleLysGly
100105110
ValGluSerGluPheTyrLeuAlaMetAsnLysGluGlyLysLeuTyr
115120125
AlaLysLysGluCysAsnGluAspCysAsnPheLysGluLeuIleLeu
130135140
GluAsnHisTyrAsnThrTyrAlaSerAlaLysTrpThrHisAsnGly
145150155160
GlyGluMetPheValAlaLeuAsnGlnLysGlyIleProValArgGly
165170175
LysLysThrLysLysGluGlnLysThrAlaHisPheLeuProMetAla
180185190
IleThr
__________________________________________________________________________
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